Protein phosphorylation is an essential and pervasive mechanism for the regulation of cellular and organismal function in nature. However for many individual phosphorylation events occurring in vivo, the technical difficulty of simultaneously identifying both the particular protein phosphorylated and the phosphorylating kinase has been a major impediment to understanding their unique physiological roles. This project will design, and optimize the technical aspects of, a method to screen for the intracellular substrates of protein kinases. This method is anticipated to have broad applicability to the study of cellular signaling processes. The principle of the method is to first map by 2-D gel electrophoresis the pattern of proteins phosphorylated in vivo in response to stimuli known to activate a particular class of protein kinases; and then to identify proteins, the stimuli-specific phosphorylation of which, is diminished by intracellular depletion of a particular kinase within that class. The Specific Aims are: Specific Aim 1 To document the presence and develop methods for relative quantification of, the kinases to be tested, CaMKIalpha and CaMKIV in a neuroblastoma cell line (BE(2)C) and in cultured primary cortical neurons. Specific Aim 2 To design small interfering RNAs (siRNAs) and test their effectiveness in knocking down individual CaMKs in these cells. Specific Aim 3 To map the patterns by 2-D gel electrophoresis of protein phosphorylation in these cells by in vivo 32p-labeling and/or fluorescent phosphoprotein staining and evaluate these patterns in response to agonists which elevate the concentration of intracellular calcium (Ca2+i). Specific Aim 4 To identify by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/MS) and peptide mass fingerprint database search, proteins, the Ca2+i-dependent phosphorylation of which is inhibited by specific CaMK knockdowns. [unreadable] [unreadable]